The endoderm gives rise to many different cell types including those that will form the liver and pancreas. How cells differentiate during embryonic development is an important focus for the field of regenerative medicine. Understanding the normal development of liver and pancreatic cell types may allow us to develop strategies for the production of hepatocytes and pancreatic beta-cells for therapeutic purposes. One potential method of producing cells for therapeutic purposes is via transdifferentiation, or, the conversion of one cell type to another. In this thesis we aimed to establish a protocol for the transdifferentiation of liver ductal cells (termed cholangiocytes) to either hepatocyte or pancreatic lineages. We also aimed to investigate the signalling pathways important for normal differentiation of embryonic liver and pancreas.In order to address the potential of a cholangiocyte cell line (biliary epithelial cells or BECs) to transdifferentiate to other cell types, BECs were infected with a combination of candidate transcription factors known as ‘master switch’ genes that have previously been demonstrated to induce transdifferentiation to hepatic or pancreatic lineages. We demonstrated that overexpression of the hepatic transcription factors C/EBPα, C/EBPβ and HNF4 resulted in the up-regulation of the hepatocyte genes Albumin and Gs and de novo expression of Afp. In complementary experiments we also demonstrated that overexpression of the pancreatic transcription factors Pdx1, Ngn3, NeuroD and Pax4 resulted in de novo expression of insulin II in BECs. While these results were encouraging further work is necessary to enhance the maturation status of the nascent cells.We also addressed the role of the Notch signalling pathway in the differentiation of embryonic hepatic and pancreatic cells using ex vivo organ culture models of liver and pancreas development. We treated pancreata with N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S- phenylglycine t-Butyl Ester (DAPT) a gamma-secretase inhibitor. Treatment with DAPT inhibits the Notch signalling pathway. Following treatment with DAPT we observed reduced branching morphogenesis, loss of the acinar cell phenotype (amylase expression) and an enhancement in endocrine differentiation (insulin and glucagon expression). We propose that in the absence of Notch signalling the proendocrine gene Ngn3 is no longer repressed by the Notch target Hes1, allowing endocrine differentiation to take place. Finally we observed that β-cells in pancreata treated with DAPT are functionally more mature in terms of responsiveness to glucose stimulation. Overall these results have important implications for the development of potential therapies in the treatment of liver failure and diabetes.
|Date of Award||1 Nov 2013|
|Supervisor||David Tosh (Supervisor)|